Synaptic transmission depends on the transport of classical neurotransmitters from the cytoplasm, where they are synthesized and accumulate after reuptake from the extracellular space, into synaptic vesicles, which then undergo regulated exocytosis. To understand how transport into synaptic vesicles contributes to neurotransmitter release, the postsynaptic response and ultimately behavior, we have focused on the proteins responsible for this basic function of the nerve terminal. In previous work, we have identified two families of proteins responsible for the transport of cationic and neutral transmitters into synaptic vesicles. However, the proteins responsible for vesicular glutamate transport have proven elusive. We have recently found that a protein previously suggested to mediate the Na+-dependent uptake of inorganic phosphate across the plasma membrane, in fact transports glutamate into synaptic vesicles. In addition to H+-driven glutamate transport, the protein (VGLUT1) exhibits an unusual chloride conductance inhibited by glutamate. To assess its channel-like properties and its transport function, we will characterize the interactions of VGLUT1 with chloride and protons. We will also assess the potential for VGLUT1 to transport glutamate (and phosphate) at the plasma membrane. Since the regulation of VGLUT1 may influence synaptic vesicle filling and VGLUT1 contains two polyproline domains, we will further characterize its interaction with other proteins. The expression of VGLUT1 by only a subset of glutamate neurons also suggests the existence of another isoform, and recent work describes a closely related putative phosphate transporter. We will thus examine the distribution of this protein and study its transport function. To assess the role of VGLUT1 in vivo, we will disrupt the gene in mice and examine the physiological effects. These experiments will help to understand the transport of glutamate at the nerve terminal and its role in transmitter release, synaptic plasticity, excitotoxicity and behavior.